The goal of this proposal is to determine how "pioneer" transcription factors, the first to bind genes in development, engage target sites in silent, compacted chromatin and promote entry of subsequent proteins. These initial steps are among the most decisive in eukaryotic gene control, yet little is known about the underlying mechanisms. Previously we showed that chromatin sites for the transcription factors HNF3 and GATA-4 are engaged in undifferentiated endoderm, prior to the activation of the albumin gene during liver specification. In the past grant period, we discovered that HNF3 and GATA-4, but not various other DNA binding factors, can bind their target sites within highly compacted chromatin in vitro and make the local nucleosomal domain accessible to other proteins. These activities do not require ATP-dependent nucleosome remodeling complexes. We further discovered a protein domain on HNF3alpha that interacts with a subset of core histones and is critical for loosening inter-nucleosomal interactions, thereby "decompacting" chromatin. In the next grant period, we will combine biochemical and transgenic approaches to understand how HNF3 and GATA factors initiate regulatory events at target sites in chromatin, with the following specific aims:1. To determine how chromatin binding by HNF3alpha and GATA factors disrupts inter-nucleosomal interactions and creates a locally open domain in defined chromatin templates in vitro.2. To determine how wild type and mutant HNF3alpha and GATA-4, when inducibly expressed in transgenic mice, open chromatin in a diverse array of silent chromatin contexts.3. To determine how HNF3 and GATA-4 binding leads to linker histone displacement, changes in nucleosome linker DNA, arid the recruitment of accessory proteins that help activate transcription.Understanding how chromatin structure is modified during the earliest stages of gene regulation will provide fundamental insight into genetic processes that are essential for growth, development, and pathogenesis in humans.